1. Technical Field
The present disclosure generally relates to methods and compositions for the synthesis of biopolymers, in particular natural or non-natural nucleic acids and analogous polymers.
2. Related Art
DNA and RNA oligonucleotides, and their structural analogs, have found numerous applications as diagnostic and therapeutic agents. Nucleic acids are the polymers used by living organisms for the storage of genetic information, and are thus a primary therapeutic target for the treatment of genetic and acquired diseases, and for vaccine development. During the past decade, interest in anti-sense and anti-gene therapy has motivated the development of DNA and RNA molecules that bind at specific points within the human genome or to specific messenger RNA sequences. More recently, the discovery of small RNA molecules that naturally interfere with gene expression has presented additional possibilities for how RNA may also be used to treat acquired and genetic diseases. Thus, methods to prepare RNA, DNA, and polymers in large scale are of great importance to the medical field for drug development and will soon be for use as therapeutics.
Living organisms, including humans, have developed protective mechanisms to degrade foreign nucleic acids that gain entry to the cells or the intercellular fluid of the organism. Such foreign, or exogenous, nucleic acids include RNA and DNA polymers introduced by naturally-occurring viruses. These same anti-pathogen defense mechanisms, however, also limit the lifetime of nucleic acid polymers introduced into a human body for therapeutic purposes. Thus, it is of great importance to develop methods for the improved synthesis of non-natural structural analogs of RNA and DNA that can function in similar capacities as natural nucleic acid polymers, but that are not susceptible to the same modes of enzymatic degradation. Structural analogs of the natural nucleic acids (or “non-natural” nucleic acids) include, but are not limited to, DNA and RNA-like polymers in which the natural backbone has been modified by the addition or removal of one or more chemical groups. These modifications range from the methylation of the ribose sugar, to the radical replacement of the backbone with a peptide-like backbone. Broadly defined, nucleic acids analogs, or non-natural nucleic acids, are any polymers that have chemical appendages to the polymer backbone similar in chemical structure to the natural nucleotide bases (i.e. A, adenine; C, cytosine; G, guanine; U, uridine; T, thymine) that allow the formation of base pairs for the transfer of nucleotide sequence information. Although the synthesis of natural nucleic acid polymers has become routine in many commercial and academic laboratories, the synthesis of non-natural nucleic acid polymers (i.e., synthetic analogs) is typically much more expensive and can require a substantial investment of time and resources for the development of a synthetic procedure for each new variation in nucleic acid chemical structure. Thus, methods that can facilitate the synthesis of natural and non-natural nucleic acids are of great economic and medicinal importance.
RNA and DNA molecules with a wide range of novel properties, including catalytic, molecular recognition and therapeutic activities, have been isolated from pools of RNA and DNA polymers, respectively, that initially contain a large number of random nucleotide sequences. This process is accomplished by multiple rounds of activity-based selection for nucleic acid molecules with a desired property from the pool of random sequences, followed by the enzymatic amplification of selected sequences using naturally-derived polymerases. Known as “in vitro selection” or “SELEX”, this procedure represents one of the most powerful forms of combinatorial chemistry.
The enzymatic amplification of nucleic acid sequences during each cycle of the SELEX procedure is an essential component of this procedure, as nucleic acid amplification leads to the exponential enrichment of sequences in a sample that have the desired molecular properties. The polymerases used to amplify nucleic acid molecules as part of the SELEX process were initially isolated from living organisms, and are still produced within host cells by fermentation. The amino acid sequences of these polymerases are either identical to that derived from a natural source, or are a genetically-modified variant of a natural polymerase. Natural polymerases or derivatives thereof cannot synthesize non-natural nucleic acids.
A process similar to SELEX for the selective amplification of non-natural RNA-like polymers would undoubtedly produce molecules superior to RNA for many applications. For example, an RNA-like polymer with a backbone that is resistant to cleavage and degradation by natural ribonucleases could be far more effective as an antibiotic because it would have a longer active time within the body. Another example would be the selection of a catalytic RNA-like polymer with a non-natural backbone that has charged groups with a pKa near neutrality. Such a catalyst would be sensitive to pH, and thus small changes in pH could be used to regulate catalytic activity. However, because naturally-derived nucleic acid polymerases can only replicate RNA or DNA, the realization of in vitro selection with non-natural polymers will either require extensive evolution of existing polymerases, or the development of new methods for molecular replication. An editorial on the SELEX procedure by a pioneer of this technique clearly states that the ability to accomplish in vitro selection using naturally-derived polymerases comes with the severe limitation that selection can only be carried out with natural polymers (Szostak, 1997). Thus, it is well appreciated by experts in this field that methods to replicate non-natural polymers would greatly expand the power of in vitro selection. Accordingly, the development of improved methods for the enzyme-free synthesis of nucleic acids and analogous polymers could be of great value to the fields of polymer chemistry, materials science, biotechnology and medicine.
Those skilled in the art of nucleic acid synthesis have toiled for decades to improve methods for enzyme-free template-directed synthesis of nucleic acids with only modest gains. For example, considerable effort has focused on the potential for inorganic cations to improve existing methods for the chemical coupling of nucleic acids in aqueous solution (Lohrmann et al, 1980; Rohatgi et al., 1996). In contrast, virtually no studies have investigated the possibility that a specific interaction between a small molecule and the nucleic acid bases could be used to enhance enzyme-free synthesis of nucleic acid polymers.
Thus, the collective body of research conducted by the scientific and medical communities demonstrates a need for more efficient methods for the chemical ligation of both natural and non-natural oligonucleotides in aqueous solution. Furthermore, there is a need for methods to efficiently couple nucleic acid polymers in solution using catalysts that are easily separated from the product molecules, so that the products can subsequently be used in therapeutic applications. An efficient, enzyme-free method for the coupling of both natural and non-natural nucleic acid oligonucleotides along a template strand would have substantial commercial value, as such methods will enable the in vitro selection of non-natural nucleic acid polymers with desirable therapeutic and catalytic properties.